Bacterial pathogenicity and ability to survive in adverse conditions depends on bacterial stress response. This proposal aims at a structural understanding of two bacterial stress- response processes, in which stalled non-translating ribosomes are being sensed: 1) stringent response, which is mediated by stringent factor RelA; and 2) rescue of stalled ribosomes by a peptidyl- tRNA hydrolase YaeJ. Bacteria adapt to insufficient nutritional conditions via a mechanism termed the stringent response. One of the consequences of nutrient deprivation is amino acid starvation, which may lead to more than a 5-fold increase in cellular levels of uncharged (deacylated) tRNAs. Deacylated tRNAs cannot participate in protein synthesis but can bind to ribosomes, which are in a paused translational state due to insufficient levels of aminoacylated tRNAs. Such stalled ribosomes are thought to interact with RelA and initiate the stringent response. RelA is an 84 kDa enzyme, which, upon binding to stalled ribosomes, catalyzes the synthesis of the small molecule alarmones ppGpp and pppGpp. These molecules trigger the stringent response by initiating a global gene expression program. The molecular mechanism of the RelA-mediated stringent response is poorly understood. First, the binding site for RelA on the ribosome has not been identified. Second, it is not known how the presence of deacylated tRNAs on the ribosome triggers the (p)ppGpp-synthesizing activity of RelA. In Specific Aim 1, we propose to address these questions by obtaining structural and dynamics information on 70S*RelA ribosome complexes. In addition to nutrient-deprivation conditions, other cellular conditions exist that result in mRNA degradation or modification, interfere with aminoacyl-tRNA binding to the A site, tRNA translocation or other steps of translation elongation. This leads to the stalling of translating ribosomes. In this stalled state, peptidyl-tRNA is stably bound to the ribosomal P site, and the ribosome is not available for initiation of translation on a new mRNA. Because ribosome synthesis requires large amounts of cell resources, it is essential that non-translating ribosomes be recycled and not degraded. To rescue such ribosomes, the incomplete protein chains and tRNAs have to be released from the ribosomes. At least two mechanisms exist, namely the well-characterized tmRNA-assisted ribosome rescue and a recently proposed YaeJ-mediated peptide release. YaeJ is a 16 kDa protein that is hypothesized to directly catalyze peptidyl-tRNA hydrolysis on the ribosome in a codon-independent manner. Our Specific Aim 2 is designed to address mechanistic questions concerning YaeJ-mediated response to ribosome stalling. The proposed aims will be accomplished by structural and biochemical methods.